This invention relates to a genetically-engineered anaerobic organism which, under anaerobic conditions present in a solid tumor, produces an enzyme capable of catalyzing the conversion of a prodrug to its highly cytotoxic product in situ and methods of treating tumors using same.
Despite the success of modern chemotherapy in curing certain types of leukemia and lymphoma and a few other relatively rare cancers, few of the current anticancer drugs have much useful clinical activity against the most common forms of cancer. These poorly responsive cancers are typically solid tumors comprising both proliferating and nonproliferating cells. Typically, the anticancer drugs used today are effective predominantly against rapidly proliferating tumor cells, and are toxic to rapidly proliferating normal tissues. Two general approaches are currently being pursued to overcome this problem. First, there is an intensive search for new drugs with selectivity against individual types of cancer cells. For example, the National Cancer Institute is currently screening some 20,000 compounds per year against a panel of 60 human tumor cells lines (Boyd, M. R. xe2x80x9cThe Future of New Drug Development,xe2x80x9d In: CURRENT THERAPY IN ONCOLOGY pp 11-22 (B. C. Decker, Inc., Neiderhuber, J. E., eds. Philadelphia 1992)). A second approach is that of targeting new or,existing drugs specifically to the tumor. Many of these approaches rely on monoclonal antibodies (Mabs) to carry a drug or toxin to the tumor. A major problem confronting most of these strategies, however, is tumor cell heterogeneity and inability to deliver the antibody conjugates to every tumor cell (Jain, R. K., (1989) J. Natl. Cancer Inst. 81:570-576). One way of overcoming this problem is the use of enzyme-antibody conjugates which activate prodrugs to form diffusible cytotoxins (Bagshawe, K. D., (1989) Br. J. Cancer 60:275-281). This approach has been called antibody-directed enzyme prodrug therapy (ADEPT). The two-step ADEPT process requires the conjugation of a suitable enzyme to a monoclonal antibody which localizes the enzyme to the tumor. When most of the nonbound antibody-enzyme conjugate has been cleared, a prodrug is administered which can be activated by the enzyme to a cytotoxic species (Bagshawe, K. D., supra; Senter, P. D. (1990) FASEB J. 4:188-193). Though the ADEPT strategy is promising, it has a number of problems. First, the large majority of the MAb-enzyme conjugates do not localize in the tumor, and studies have shown that concentrations of the active drug in normal tissues can be greater than in the tumor (Antoniw et al., (1990) Br. J. Cancer 62:909-914). Also, MAbs of high enough specificity are not available for many tumors.
Other targeting approaches include the use of recombinant toxins, such as growth factors fused to a bacterial toxin (Fitzgerald et al., (1992) Biochem. Soc. Trans. 20:731-734), and the use of tumor-infiltrating lymphocytes genetically engineered to produce a protein such as tumor necrosis factor (TNF) (Rosenberg, S. A., (1992) JAMA 268:2416-2419). No reports of improved activity of any of these targeting strategies has yet appeared.
In another approach, a gene for the cancerostatic polypeptide Colicin E3 was introduced into the uncharacterized mixture of endogenous plasmids in C. oncolyticum (C. butyricum M-55, renamed because of its oncolytic activity) (Schlechte, H, and B. Albe, (1988) Zbl. Bakt. Hyg. A 268:347-356). This approach, however, was unsuccessful. They were unable to show that these recombinants were expressing active protein. Though this approach might ultimately lead to a clostridial strain expressing an anticancer polypeptide, the major drawbacks of this strategy are first, that the anticancer agent must be a protein rather than a low molecular weight anticancer drug which presently constitute all chemotherapeutic agents, and, second, the inability to control the production of the toxic product.
Accordingly, a need exists for a means for selectively targeting a toxic chemotherapeutic agent to solid tumor tissue without exposing healthy tissue to the agent.
This invention is based on the finding that an anaerobic microorganism can be genetically engineered to produce an enzyme that can catalyze the conversion of a non toxic prodrug to a highly cytotoxic chemotherapeutic agent. When injected into a subject, the genetically engineered microorganism will proliferate and produce the enzyme exclusively in the hypoxic/necrotic regions of a tumor in an otherwise healthy individual. The tumor-bearing individual is treated systemically with a non toxic prodrug which is converted to the highly toxic counterpart by the enzyme which, due to the anaerobic nature of the microorganism producing it, will only be present in the hypoxic/necrotic regions of the tumor. Since the enzyme is localized to the tumor, the generation of the toxic product is also localized to the tumor thus preventing the systemic toxicity associated with direct administration of the toxic chemotherapeutic agent but providing for the diffusion of the enzyme, prodrug and toxic product throughout the tumor.
Accordingly, one aspect of the invention is a vector expressed in obligate anaerobes for the production of an enzyme capable of converting a non toxic prodrug to a toxic chemotherapeutic agent. A member of the genus Clostridium is a preferred anaerobe. Clostridium acetobutylicum is a particularly preferred anaerobe. The enzymes nitroreductase, B-glucuronidase and cytosine deaminase are preferred enzymes. A clostridial vector comprising the ntr gene encoding E. coli B nitroreductase. (NTR) and the promoter and RBS of the ferredoxin (Fd) gene of Clostridium pasteurianum for the expression of nitroreductase in Clostridium acetobutylicum is a preferred embodiment of the present invention. CB1954, 5-fluorocytosine, and glucuronides of epirubicin, 5-fluorouracil and 4-hydroxycyclophosphamide are preferred prodrugs.
Another aspect of the invention is a method of targeting a toxic chemotherapeutic agent to a tumor in a tumor-bearing individual comprising the steps of:
a) administering an effective amount of a genetically engineered anaerobic microorganism capable of proliferating and producing an enzyme in the hypoxic/necrotic environment of a tumor to said individual; and then
b) systemically administering a prodrug which is converted at the site of the tumor to the toxic chemotherapeutic agent by the enzyme produced by the microorganism. A method comprising administering an effective amount of Clostridium acetobutylicum genetically engineered to produce E. coli B nitroreductase (NTR) and then administering the prodrug CB1954 is a preferred method. Other preferred methods involve administering Clostridium acetobutylicum genetically engineered to produce xcex2-glucuronidase or cytosine deaminase and then administering prodrugs comprising glucuronides of epirubicin, 5-fluorouracil, and 4-hydroxycyclophosphamide, or 5-fluorocytosine, respectively.